The ability to effectively click-label a target molecule is dependent on the reactive groups present on both the label and target molecule in the reaction and the conjugation conditions. Reagents such as succinimidyl esters (SE) and perfluorophenyl (PFP) esters have high reactivity rates with water, thereby limiting preparation, packaging, dispensing and purification conditions and their subsequent shelf life. In addition, due to their hydrolytic reactivity, most of the reagents used in a biomolecule labeling reaction in aqueous buffers hydrolyze prior to reaction with the desired biomolecule; therefore, such reagents are largely wasted (often necessitating their use in molar excess).
Gee et al. (Tetrahedron Letters (1999), 40, 1471-1474) describes 4-sulfotetrafluorophenyl (STP) esters for use in dye labeling. These groups have been shown to be amenable to conjugation in aqueous environments.
Koichi et al. (Chemical & Pharmaceutical Bulletin (1987), 35(3), 1044-1048) and Tsuji et al. (Peptide Chemistry (1986), Volume Date 1985, 23rd 111-114) describe peptide synthesis via ester activation with potassium dichlorophenolsulfonate, sodium dibromophenolsulfonate, and sodium nitrophenolsulfonate. No description of labeling or conjugation of molecules is provided.
While many labeling reagents exist and have been used with intermittent success, there remains a need for click-labeling reagents that produce high labeling yields under biologically relevant reaction conditions. Additionally, a need exists for click-labeling reagents that are stable and do not hydrolyze in aqueous environments.